ADP-ribosylation is a post-translational protein modification synthesized by a family of 17 enzymes, called PARPs (poly(ADP-ribose) polymerases). Human cells express 17 different PARP proteins; the functions and modification targets of many have not yet been identified. Deeper understanding of the PARP family is clinically important, as PARPs are highly druggable enzymes and PARP inhibitors directed against PARP-1, the founding member of the PARP family, are currently in over 14 clinical trials for the treatments of breast, ovarian, lung, skin and colorectal cancers. The Macro subfamily of PARPs (PARP-9, -14 and -15) was initially identified in a screen for proteins whose over-expression in B-cell lymphoma cells resulted in increased cell migration rates, and PARP-9 and PARP-14 have since been linked to roles in cancer cell migration. While these early analyses suggested functions for the Macro PARPs in the regulation of cell motility, the mechanism of this regulation has remained unclear. Recently, we have found that depletion of either PARP-9 or PARP-14 from human somatic cells causes severe defects in cell motility and membrane dynamics, along with defects in the activity of a small GTPase required for proper cell motility, RhoA. The objective of the proposed research is to determine both the targets and the mechanisms of PARP-9 and PARP-14 activity that contribute to the regulation of RhoA signaling and cell motility. The first proposed experimental aim tests the hypothesis that PARP-9 and -14 directly contribute to the proper spatiotemporal control of RhoA activity in human cells. To this end, the localization and activity state of RhoA in actively migrating cells will be examined using FRET-based reporters, and the effects of PARP-9 and PARP-14 depletion on this spatiotemporal activity will be tested. The localization of PARP-9 and -14 will also be observed, in real time, in relation to sites of RhoA activity. Finally, it will be directly tested if RhoA isa direct target of ADP-ribosylation, and if PARP-9 or PARP-14 contributes to this modification. The second experimental aim will determine the activity and the domains of PARP-9 and PARP-14 required for proper cell motility and membrane dynamics, by testing the ability of various PARP-9 and PARP-14 mutants to rescue the observed depletion phenotypes. The third aim will take a biochemical approach to identify PARP-9 and -14 binding partners and ADP-ribosylation targets that may contribute to cell motility and RhoA regulation. How ADP-ribose modification of potential target proteins contributes to cell motility and membrane dynamics will also be explored. Together, these experiments will further our understanding of PARP protein function in human cells and will describe a novel role for PARPs in cell migration and RhoA regulation. Importantly, deregulated cell motility contributes to metastasis, and small molecule inhibitors targeting the cell motility machinery decrease invasion and suppress tumor growth. Thus in the future, drugs targeting PARP-9 and PARP-14 functions in cell motility may be promising therapeutic treatments for metastatic cancers. PUBLIC HEALTH RELEVANCE: Many physiological processes, including wound closure, embryonic development and immune responses, require individual cells within the body to rapidly and responsively migrate from one location to another. Defects in this controlled migratory behavior can contribute to the development of metastatic cancers; understanding the proteins and the signals involved in cell migration is therefore critical to understanding both normal and disease processes. The research training program described in this application will explore and define a novel role for two proteins, PARP-9 and PARP-14, in the control of cell migration, thereby extending our understanding of how cells direct their movements, and elucidating two proteins that can be targeted to stop the spread of metastatic cancer cells.